Inhibition of retrovirus-encoded enzymes may offer a viable option to interrupt the replication and propagation of these viruses. An intense investigation on the mechanism of inhibition of reverse transcriptase led to the discovery of 3-azido-3, deoxythymidine (AZT), an inhibitor of this enzyme, as a chemotherapeutic agent for the patients infected with the human immunodeficiency virus (HIV-1). There is now growing interest in another enzyme encoded by the HIV-1 genome, the aspartic protease that processes the viral polypeptides into mature virion proteins. A potent and specific inhibitor of this enzyme may serve as a novel therapeutic agent. In order to generate specific oligopeptides derived from the HIV-1 aspartic protease antibody-structure we have generated monoclonal antibodies directed against an N-terminal and a C-terminal peptide from HIV-1 aspartic protease. Three hydridomas have so far been characterized which produces antibodies that recognize HIV-1 AP as determined by Western blotting. To determine whether these antibodies inhibit the protease activity we developed a simple and specific radiometric assay to screen the inhibitory activity of these antibodies. In this assay an oligopeptide was synthesized, radiolabeled and used as the substrate. The proline residue was substituted by the dehydroproline during peptide synthesis and in an exchange reaction it was tritiated. Because of the hydrophobicity of this oligopeptide it avidly binds to dextran-coated activated charcoal tablets. The Ap cleaves this peptide into two fragments. The intact peptide is very hydrophobic and is absorbed by dextran-coated charcoal; the relatively hydrophilic peptide fragment generated as a result of AP catalysis dissolves in the supernatant. Since the 3H-labeled amino acid is present in the hydrophilic portion of the molecule, the product is quantitated by simply counting aliquots of the supernatant in a beta counter. This assay is now being standardized and will be used in testing the monoclonal antibodies raised against the specific regions of HIV-1 AP for their inhibitory activity.